Overproduction of IL-12 is associated with autoimmune diseases and microbial infections. Newly synthesized leaderless proIL-12 cannot be efficiently secreted from activated monocytes. However, when LPS-primed monocytes are further stimulated with extracellular ATP, they rapidly release large amounts of mature IL-12 and proIL-12. Thus far, the mechanism by which IL-12 is released from human monocytes is not understood. The overall goal of this proposal is to determine how IL-12 is released by investigating the molecular mechanisms by which ?-defensins block the release of IL-12 from human monocytes. Human ?-defensins (HNP-1 and HD-5), a group of antimicrobial peptides produced by neutrophils and epithelial cells, are the only inhibitors that block the release but not the processing of proIL-12 by caspase-1 in human monocytes. This proposal does not investigate the in vivo role of defensins. It only uses defensins as a tool to identify novel molecular targets for IL-12 blockade. Using photo-affinity and confocal microscopy, we have found that defensins bind to cell-membrane-associated proteins in human monocytes. We will test the hypothesis that IL-12 release and the externalization of proIL-12 and secretory lysosomes from human monocytes are regulated by distinct signaling proteins which can be inhibited by ?-defensins via an enzyme- linked receptor. To accomplish that, we have identified two specific aims: Specific Aim One: Identify the receptor that is responsible for defensin-mediated inhibition of IL-12 release from human monocytes. Using photo-affinity purification and proteomic approaches, we will identify defensin-binding proteins (DBPs) in human monocytes. We will then identify the defensin receptor by determining the effect of siRNA-mediated knockdown of these DBPs on defensin blockade of IL-12 release. Specific Aim Two: Define the signaling pathway essential for defensin receptor-mediated inhibition of IL- 12 release. We will determine whether protein kinase C, phospholipase C, Ca2????dependent protein kinase, and MAP kinases are involved in defensin blockade of IL-12 release and the externalization of proIL-12 and secretory lysosomes from human monocytes. Successful completion of this project will have broad impacts on both basic and translational medical research. If our hypotheses are correct, it will not only bring much clarity to the field of IL-12 biology, but also provide novel insights to the ER/Golgi-independent secretory pathway used by many other important leaderless proteins, including IL-18, IL-33, MIF, and FGF-2. The proposed studies will also lead to the discovery of novel molecular targets for IL-12 blockade and may lead to the development of new therapeutic approaches to prevent and treat many life-threatening microbial infections and inflammatory diseases. PUBLIC HEALTH RELEVANCE: Defensins are peptides produced by white blood cells and epithelial cells. Using defensins as a tool, we will determine how proinflammatory cytokine IL-12 is released from human monocytes. The proposed studies will lead to the discovery of novel molecular targets for IL-12 blockade and may lead to the development of new therapeutic approaches to prevent and treat many life-threatening microbial infections and inflammatory diseases.